The present invention relates to a method of selectively analyzing structural isomers of dicarboxylic acids based on the unique fragmentation of their derivatives using mass spectrometry (MS), and enhancement of selectivity for the analysis of the dicarboxylic acids through choice of the utilized derivative. In particular, the present invention relates to the determination of methylmalonic acid in a biological sample, and to the diagnosis of vitamin B12 deficiency.
Measurement of methylmalonic acid (MMA) became an important diagnostic procedure in clinical chemistry due to accumulated evidence that slightly increased concentrations of MMA was a marker of vitamin B12 deficiency. MMA is a metabolic intermediate in the conversion of propionic acid to succinic acid (SA). Vitamin B12 is an essential cofactor of the enzymatic carbon rearrangement of MMA to succinic acid and the lack of vitamin B12 leads to elevated levels of MMA. Elevated levels of methylmalonic acid were found in the urine of vitamin B12-deficient patients [1]. Deficiency of vitamin B12 also causes serious and often irreversible neurological disorders named as subacute combined degeneration of the spinal cord [2]. A moderately elevated concentration of MMA (above 0.4 xcexcmol/L in serum or plasma and above 3.6 mmol/mol CRT in urine) is an early indicator of vitamin B12 deficiency. Frequency of positively tested samples with results consistent with vitamin B12 deficiency is 1:20 to 1:50 depending on the population tested. Massive elevation of MMA in serum, plasma or urine (100 to 1000 fold above the concentrations characteristic for the vitamin B12 deficiency) is consistent with methylmalonic acedemia, an inborn metabolic disorder. Frequency of the methylmalonic acedemia disorder in newborns is 1:20,000 [1,3].
Although, serum MMA and serum cobalamin measurements seem to have equal clinical sensitivity in detecting vitamin B12 deficiency, there are advantages of measuring MMA instead of cobalamin. Firstly, serum or plasma vitamin B12 levels may not reflect adequately tissue cobalamin status. Secondly, serum MMA level is 1000-fold greater than serum cobalamin level, and an elevation rather than decreased concentration is measured in vitamin B12 deficiency. Thirdly, MMA is more stable than cobalamin.
Since the 1960s efforts have been directed towards developing a rapid, simple, sensitive, and specific analytical method for methylmalonic acid determination in biological fluids. In general, sample preparation is required which consists of MMA extraction step from a sample matrix, and, most of the time, a subsequent derivatization. To be able to detect vitamin B12 deficiency the method is required to measure the low concentrations of MMA found in urine and serum (xcx9c1 xcexcmol/L in urine, xcx9c0.1 xcexcmol/L in serum). The derivatization step is necessary to improve MMA detection by UV or fluorescent detector [4-10], or to convert the organic acid to a volatile derivative for GC separation [11-30].
Solvent [7,10,12,18-20,22-27,33-36,38,39] and solid-phase extractions [9,11,16,28,29,31,37], preparative chromatography [7,13] or solvent extraction and HPLC (combined) [14,17] have been used to separate MMA from biological samples prior to an instrumental analysis. For serum specimens, a protein precipitation step precedes the extraction. For solvent extraction, the preferred solvents have been diethyl ether, ethylacetate, or both. High MMA recovery was required otherwise the analytical method was not sensitive enough to detect MMA. Some authors used multiple extraction and combined the extracts [10,12,19,20,22-24,34-36], while others utilized saturated NaCl to increase ionic strength of the solution [19,20,22-25,34]. In some cases the extracts were dried with MgSO4 or Na2SO4 in order to eliminate residual water for the subsequent derivatization for GC analysis [10,12,14,20]. Generally, tedious extraction was required to reduce possible interferences and to obtain an extract that was suitable for further analysis.
There are some methods described in the literature which do not include an extraction step [4-6,8]. Among these are paper [4] and thin-layer chromatography [6,8], colorimetry [5], GC-MS [15], and LC-MS [31,32]. Paper and thin-layer chromatography were used only as qualitative screening methods [4,6,8], and positive specimens were subjected to the more specific and quantitative GC or GC-MS analysis [8]. Norman et al. [15] did not use extraction for urine dicarboxylic acid determination. After addition of the internal standard solution, the sample was evaporated to dryness and derivatized for subsequent GC analysis. This method can be used to identify inborn errors of metabolism from urine samples only and cannot be applied to serum specimens because of their high protein content. The two LC-MS based methods [31,32] were developed for urine organic acid analysis in inborn errors of metabolism screening, and were not optimal for determination of even mildly elevated concentrations of MMA. The authors were able to see methylmalonic acid only at very elevated levels. Instrumental analysis time, using any of the methods described above, range from 10-60 minutes per sample. Furthermore, these methods were only suitable to identify patients with methylmalonic acedemia and were not sensitive enough for the vitamin B12 deficiency screening.
Derivatization schemes that have been used in methods of determining MMA are method dependent In TLC, HPLC, or CE, derivatization of MMA is required for detection purposes. In GC methods, derivatization is required to convert MMA to a volatile derivative. There are few published methods where analysis of MMA did not require derivatization [31,32,34-39]. Mills et al. [31], Buchanan et al, [32], Kajita et al. [37] have used LC-MS to analyze organic acids in urine specimens No derivatization was needed, however, none of these methods were sensitive enough to analyze MMA in normal urine specimens. Frenkel et al. [34] describe a GC method for urinary MMA determination without derivatization of MMA. MMA from urine specimens was extracted and directly injected into a GC. At the injection port temperature of 225xc2x0 C. MMA decomposed to propionic acid, and was analyzed as such. This result gave the sum of propionic and methylmalonic acid in the specimen. From a second injection with a lower injection port temperature, propionic acid alone was determined. Concentration of MMA was calculated as the difference between the two measurements. Mikasa et al. [35] describe an isotachophoresis method for urine MMA determination which included an extraction but no derivatization step. Although the detection limit using this method was 0.4 xcexcmol/L MMA in urine samples, it was achieved by extracting 10 mL of urine. This method is clearly not practical and sensitive enough for serum specimens. Rinaldo et al. [36] describe a CAD MIKES (collisionally activated decomposition mass analyzed kinetic energy spectrometry) technique which has been used to identify patients with methylmalonic acedemia. The technique is not quantitative and by no means is sensitive enough to measure normal concentrations of MMA in urine or serum. Nuttall et al. [38,39] reported a capillary electrophoresis method for MMA determination in urine [38] and serum [39]. To avoid derivatization, they used indirect UV detection; however, using this method, sensitivity was limited and there was no specificity. All the above methods were designed to diagnose organic acedemias in urine specimens. Accordingly, none have adequate sensitivity to measure MMA in serum samples for diagnosis of vitamin B12 deficiency.
The work by Allen et. al [40] on MMA and SA analysis by GC/MS showed that the t-butylsilyl ester derivatives of the compounds gave nearly identical mass spectra showing analytically useful fragment ions at m/z 331 and 289 MMA and SA were analyzed by GC/MS using SIM of the common 289 fragment ion requiring that MMA and SA be chromatographically separated prior to MS detection. The method has sufficient sensitivity and performance characteristics to allow determination of both vitamin B12 deficiency and methylmalonic acedemia. The disadvantages associated With this GC/MS method is the requirement for time- and resource-consuming sample preparation as well as a relatively low throughput (3-6 samples per hour).
Recent work by Johnson [41] involves the determination of long- and very-long chain fatty acids by Ionspray (nebulizer assisted electrospray) MSxe2x80x94MS. The fatty acids are derivatized to form dimethylaminoethyl (DMAE) esters via a 2-step condensation reaction using oxalyl chloride and dimethylaminoethanol as reagents. The analogous reaction is the use of acylchloride and butanol (which acts like butanolic hydrochloride) to form butyl esters of amino acids and acylcamitines [42]. The DMAE esters are ionized in the positive ion mode and fragment to form a 45 u neutral loss (dimethyl amine) and strong characteristic product ions of m/z 72 and 90. This allows for several ways of screening for a wide variety of acids using neutral loss and precursor ion MSxe2x80x94MS scans. The reagent produces a strongly basic derivative which would very much enhance the response of these acids in the positive ion mode. This reagent should work for MMA and SA; however, the specificity for the selective ionization of dicarboxylic acid butyl esters over other acids would likely be lost since all the acids present in a sample would be derivatized and the amino group on the DMAE moiety would readily protonate. In other words, DMAE esters of all acids, regardless of their structure, would ionize equally well. Furthermore, the fragmentation of DMAE ester derivatives is dominated by fragmentation reactions described above and would likely not lead to a dramatic difference in fragmentation between the isobaric acids MMA and SA.
Work by Chace, et. al. [42] demonstrated the FIA (Flow Injection Analysis)-MSxe2x80x94MS analysis of amino acids as n-butylated ester derivatives. This included the amino acids Glu and Asp which have two carboxylic acid moieties. In their work, the purpose of the derivatization was to force cationic character upon these amino acids as well as eliminate interferences from other endogenous analytes which do not undergo the neutral loss of 102 MSxe2x80x94MS transition characteristic of amino acids. Isomeric amino acids still interfere with one another. Once derivatized the remaining amino group is readily protonated under eleviated pH and IonSpray [43]. The method was shown only to be effective for the analysis of amino acids and acylcamitines with no reference to the analysis of dicarboxylic acids such as MMA.
Even though esterification of acidic analytes is commonly used for GC/MS analysis there is no reference to the use of such derivatives for the selective LC/MS analysis of dicarboxylic acids and for MMA and SA in particular. The purpose of esterification of MMA for GC/MS analysis is to render the molecule sufficiently nonpolar to be amenable to GC. Since the GC/MS spectra of MMA and SA esters are nearly identical [40], GC separation of these isobaric analytes is required prior to MS analysis. For LC/MS applications employing API (atmospheric pressure ionization) techniques like Ionspray, organic acid esterification would be counterintuitive because the nonpolar derivative in solution would be less likely to be ionized than its underivatized form.
The major obstacle for MMA analysis in biological fluids is potential interference by low molecular weight organic acids, and especially from the naturally occurring structurally related isomer, succinic acid. Chromatographic characteristics and mass spectra of succinic acid are almost identical to that of MMA and because succinic acid is a product of MMA degradation and is usually present in samples at a greater concentration than MMA, succinic acid interference is difficult to overcome. There is a need, thus, for a method of determining MMA in biological samples in order that vitamin B12 deficiency and methylmalonic acedemia can effectively be diagnosed without interference from succinic acid.
Accordingly, the present invention provides a method for determining the presence of a dicarboxylic acid in a sample comprising the steps of:
extracting the acidic component from the sample;
derivatizing the acidic component; and
using mass spectromety and atmospheric pressure ionization in the positive ion mode to determine the presence of a dicarboxylic acid of interest.
In another aspect, the present invention provides a method for determining the presence and quantity of methylmalonic acid in a sample comprising the steps of
1) extracting the acidic component of the sample;
2) derivatizing the acidic component; and
3) determining the presence of methylmalonic acid using mass spectrometry and atmospheric pressure ionization in the positive ion mode.
In another aspect of the present invention, there is provided a method for diagnosing vitamin B12 deficiency in a patient comprising the steps of:
1 ) obtaining a biological sample from the patient;
2) extracting the acidic component from the sample;
3) derivatizing the acidic component;
4) analyzing the sample by mass spectrometry employing atmospheric pressure ionization in the positive ion mode; and
4) determining the presence of methylmalonic acid at a concentration of at least 0.4 xcexcmol/L in the sample.
The present invention advantageously provides a mass spectrometry method having a specificity toward the detection of dicarboxylic acids present in a sample, since all other acids present in the sample that do not have a dicarboxylic acid moiety are undetectable under the conditions of this method. The detection of dicarboxylic acids is accomplished by exploiting the unique ionization of derivatized dicarboxylic acids that occurs in the positive ion mode of an atmospheric pressure ionization (API) ion source such as IonSpray or electrospray. The method also provides a means for distinguishing between isobaric dicarboxylic acids, such as methylmalonic acid (MMA) and succinic acid (SA), by exploiting the unique collisionally induced decomposition (CID) fragmentation thereof that occurs when molecular ions of derivatized MMA and SA undergo upfront (in source) CID in single MS, or CID in a tandem mass spectrometer. As a result, the necessity for an initial separation step, to separate such isobaric components, can be eliminated thereby simplifying sample analysis and significantly reducing analysis time.